Genetic control of kinetochore-driven microtubule growth in Drosophila mitosis

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM) and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed.

First person – Chujun Zhang

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Chujun Zhang is first author on ‘ Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells’, published in JCS. Chujun is a PhD student in the lab of Prof. Catherine Rabouille at Hubrecht Institute of the KNAW and UMC Utrecht, Utrecht, The Netherlands, investigating pathways leading to stress assemblies formation.

Activation of salt Inducible Kinases, IRE1 and PERK leads to Sec bodies formation in Drosophila S2 cells

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the ER exit sites under the stress of amino-acid starvation. Here we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt inducible kinases (SIK) by Na+ stress, that when it is strong is sufficient. The second is activation of IRE1 and PERK downstream of ER stress induced by absence of amino- acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK and IRE1/PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work pioneers the role of SIK in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino-acids and salt.

RiboTRIBE: Monitoring Translation with ADAR-meditated RNA Editing

RNA translation is tightly regulated to ensure proper protein expression in cells and tissues. Translation is often assayed with biochemical assays such as ribosome profiling and TRAP, which are effective in many contexts. These assays are however not ideal with limiting amounts of biological material when it can be difficult or even impossible to make an extract with sufficient signal or sufficient signal:noise. Because of our interest in translational regulation within the few Drosophila adult circadian neurons, we fused the ADAR catalytic domain (ADARcd) to several small subunit ribosomal proteins and assayed mRNA editing in Drosophila S2 cells. The strategy is named RiboTRIBE and is analogous to a recently published APOBEC-based method. The list of RiboTRIBE-edited transcripts overlaps well with ribosome profiling targets, especially with more highly ranked targets. There is also an enriched number of editing sites in ribosome-associated mRNA comparing to total mRNA, indicating that editing occurs preferentially on polyribosome-associated transcripts. The use of cycloheximide to freeze translating ribosomes causes a substantial increase in the number of RiboTRIBE targets, which is decreased by pretreating cells with the chain terminating drug puromycin. The data taken together indicate that RiboTRIBE successfully identifies transcripts undergoing active translation.

Dynamics of Whole-Genome Contacts of Nucleoli in Drosophila Cells Suggests a Role for rDNA Genes in Global Epigenetic Regulation

Chromosomes are organized into 3D structures that are important for the regulation of gene expression and differentiation. Important role in formation of inter-chromosome contacts play rDNA clusters that make up nucleoli. In the course of differentiation, heterochromatization of rDNA units in mouse cells is coupled with the repression or activation of different genes. Furthermore, the nucleoli of human cells shape the direct contacts with genes that are involved in differentiation and cancer. Here, we identified and categorized the genes located in the regions where rDNA clusters make frequent contacts. Using a 4C approach, we demonstrate that in Drosophila S2 cells, rDNA clusters form contacts with genes that are involved in chromosome organization and differentiation. Heat shock treatment induces changes in the contacts between nucleoli and hundreds of genes controlling morphogenesis. We show that nucleoli form contacts with regions that are enriched with active or repressive histone marks and where small non-coding RNAs are mapped. These data indicate that rDNA contacts are involved in the repression and activation of gene expression and that rDNA clusters orchestrate large groups of Drosophila genes involved in differentiation.

Sec71 separates Golgi stacks in Drosophila S2 cells

ABSTRACTGolgi stacks are the basic structural units of the Golgi. Golgi stacks are separated from each other and scattered in the cytoplasm of Drosophila cells. Here, we report that the ARF-GEF inhibitor Brefeldin A (BFA) induces the formation of BFA bodies, which are aggregates of Golgi stacks, trans-Golgi networks and recycling endosomes. Recycling endosomes are located in the centers of BFA bodies, while Golgi stacks surround them on their trans sides. Live imaging of S2 cells revealed that Golgi stacks repeatedly merged and separated on their trans sides, and BFA caused successive merger by inhibiting separation, forming BFA bodies. S2 cells carrying genome-edited BFA-resistant mutant Sec71M717L did not form BFA bodies at high concentrations of BFA; S2 cells carrying genome-edited BFA-hypersensitive mutant Sec71F713Y produced BFA bodies at low concentrations of BFA. These results indicate that Sec71 is the sole BFA target for BFA body formation and controls Golgi stack separation. Finally, we showed that impairment of Sec71 in fly photoreceptors induces BFA body formation, with accumulation of both apical and basolateral cargoes, resulting in inhibition of polarized transport.

Metabolomic Analysis of Cricket paralysis virus Infection in Drosophila S2 Cells Reveals Divergent Effects on Central Carbon Metabolism as Compared with Silkworm Bm5 Cells

High-throughput approaches have opened new opportunities for understanding biological processes such as persistent virus infections, which are widespread. However, the potential of persistent infections to develop towards pathogenesis remains to be investigated, particularly with respect to the role of host metabolism. To explore the interactions between cellular metabolism and persistent/pathogenic virus infection, we performed untargeted and targeted metabolomic analysis to examine the effects of Cricket paralysis virus (CrPV, Dicistroviridae) in persistently infected silkworm Bm5 cells and acutely infected Drosophila S2 cells. Our previous study (Viruses 2019, 11, 861) established that both glucose and glutamine levels significantly increased during the persistent period of CrPV infection of Bm5 cells, while they decreased steeply during the pathogenic stages. Strikingly, in this study, an almost opposite pattern in change of metabolites was observed during different stages of acute infection of S2 cells. More specifically, a significant decrease in amino acids and carbohydrates was observed prior to pathogenesis, while their abundance significantly increased again during pathogenesis. Our study illustrates the occurrence of diametrically opposite changes in central carbon mechanisms during CrPV infection of S2 and Bm5 cells that is possibly related to the type of infection (acute or persistent) that is triggered by the virus.